Four potential TMPRSS2 protease inhibitors were tested:

- Chemostat

- Upamostat

- Nafmostat

- Bromhexine hydrochloride

Each of these existing, clinically proven drugs were found to bind to the catalytic pocket of TMPRSS2 with high precision and specificity.  Theoretically, these drugs could impede SARS-CoV-2 S proteins from interacting with TMPRSS2 which would consequently influence infectivity.  This could be a hopeful target for antiviral drugs or a future vaccine.  

Using Serine Protease Inhibitors to Treat or Prevent COVID-19

Two potential TMPRSS2 binding sites on the SARS-CoV-2 spike protein have deep identified:

- Site 1: aa837 to aa 845

- Site 2: aa 976 to aa986

Both of these sites are located in the S2 subunit of the S protein.  As shown in this figure, these binding sites are largely homologous to those of SARS-CoV-1.

TMPRSS2 Binding Sites in SARS-CoV-2 S Protein

Because the incidence and mortality rates of COVID-19 are significantly different in different regions of the world, it is thought that human variation may play a greater role than viral variation in determining these outcomes.  Significant variation in the gene TMPRSS2, which codes for the protease TMPRSS2, has been observed in different ethnic groups.  This may contribute to the different rates of infectivity seen in different populations.  

Genetic variation within the TMPRSS2 protease may play a role in COVID-19 infection

This graph displays the variance in the amount of TMPRSS2 expression there is in different types of tissues within the human body.

Graph of the Expression of TMPRSS2 in Various Tissues

- Arg and Lys residues within ACE2 amino acids 697-716 are required for cleavage
- Cleavage of ACE2 receptor might promote spike protein driven entry of the virus

TMPRSS2 Cleavage of ACE2

TMPRSS2 is a human serine protease, an enzyme that cleaves a peptide chain via hydrolysis.  This enzyme has been shown to activate SARS-Cov-2 S protein by cleaving the S1 and S2 subunits of the S protein in order to expose the S2 subunit.  This allows the viral membrane to fuse with the cellular membrane.  Consequently, this process helps facilitate infection.  TMPRSS2 is also responsible for cleaving ACE2 receptors to further enhance SARS-CoV-2 entry.  This serine protease does not have a known, relevant function in human physiology and therefore could be a promising therapeutic target.  

Michigan State University

Retrospective epidemiological studies using clinical data and experimental molecular biologies studies have revealed biomarkers of COVID-19.  These elements not only reveal how COVID-19 mechanistically affects the body, but they may also be risk factors for developing severe symptoms and act as targets for pharmacological intervention.  They include:

- A1AT protection
- ACE2 expression and activity
- ADAM17 sheddase activity
- Furin protease activity
- Irregular respiratory function
- Pro-inflammatory cytokine concentration in the plasma
- TMPRSS2 serine protease genetic variation
- Variation in genetic susceptibility to SARS-CoV-2
- Viral interactions with glycosaminoglycans
- Nsp (Non-specific Protein) Activity
- Neuropilin-1 expression


Pathogenesis of COVID-19

Transmembrane protease serine 2 (TMPRSS2) is a serine protease.  Its gene is located on chromosome 21 at 21q22.3.  Androgen signaling controls its expression upstream of the gene's transcriptional start site.  

TMPRSS2

Vishnubhotla, R., Vankadari, N., Ketavarapu, V., Amanchy, R., Avanthi, S., Bale, G., ... & Sasikala, M. (2020). Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex. bioRxiv. https://www.biorxiv.org/content/10.1101/2020.06.30.179663v1.full
doi: 10.1101/2020.06.30.179663

Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex

Hou, Y., Zhao, J., Martin, W., Kallianpur, A., Chung, M. K., Jehi, L., ... & Cheng, F. (2020). New insights into genetic susceptibility of COVID-19: an ACE2 and TMPRSS2 polymorphism analysis. BMC Medicine, 18(1), 1-8.
https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-020-01673-z

New insights into genetic susceptibility of COVID-19: an ACE2 and TMPRSS2 polymorphism analysis

de Loyola, M. B., dos Reis, T. T. A., de Oliveira, G. X. L. M., da Fonseca Palmeira, J., Argañaraz, G. A., & Argañaraz, E. R. Alpha‐1‐antitrypsin: A possible host protective factor against Covid‐19. Reviews in Medical Virology, e2157.
https://onlinelibrary.wiley.com/doi/full/10.1002/rmv.2157
doi: 10.1002/rmv.2157

Alpha‐1‐antitrypsin: A possible host protective factor against Covid‐19

TMPRSS2 in the Pathogenesis of COVID-19

SARS-CoV-2 S protein has two functional domains that regulate entry into a host cell:

- receptor binding domain (RBD)/ACE2 domain
- second domain (S2)

The S1 and S2 subunits of the S protein are connected by a polybasic furin cleavage site.  When furin acts on this site, the RBD and S2 domain are exposed which facilitate viral entry.    

Furin in the Pathogenesis of COVID-19

- European Wide Cohort Study Examines Expression Levels of COVID-19 Susceptible Biomarkers in Patients with Severe Asthma
- Review Investigating the Immunopathogenesis of COVID-19
- Structures and distributions of SARS-CoV-2 spike proteins on intact virions
- Implications of sex differences in immunity for SARS-CoV-2 pathogenesis and design of therapeutic interventions
- Natural killer cell immunotypes related to COVID-19 disease severity

Studies Related to the Pathogenesis of COVID-19

Three primary observations have been made about ACE2 and its relation to SARS/COVID-19:

- ACE2 acts as a functional receptor for SARS-CoV-2 to enter host cells
- Increased ACE2 expression may elevate the risk of a SARS-CoV-2 infection as well as the development of COVID-19
- Reduced ACE2 expression may lead to acute respiratory distress syndrome

SARS‐CoV‐2 may be able to infect other tissues that express ACE2 using viral RNA that circulates in the blood.  

ACE2 in the Pathogenesis of COVID-19

Differences in COVID-19 inter-individual variability and lethality, along with differences among countries, raises the consideration of the role that predisposed genetics may play. Genetic variation may explain why some individuals without COVID-19 risk factors develop severe COVID-19.  Functional polymorphisms in the following genes are of interest due to their coded proteins' role in the pathogenesis of COVID-19:

- ACE2
- TMPRSS2

Understanding these variations in different populations could lead to more individualized treatment in COVID-19 patients.

Genetic Susceptibility to SARS-CoV-2 Infection in the Pathogenesis of COVID-19

Many viruses, including beta coronaviruses, have been shown to interact with human glycosaminoglycans, which are negatively-charged polysaccharides.  Studies suggest that SARS-CoV-2 can bind to the glycosaminoglycans heparan sulphate and heparin.  Additionally, it is thought. that heparin can prevent the virus from entering host cells while heparan sulphates facilitate infection by keeping SARS-CoV-2 S protein in its open conformation which allows it to interact with ACE2 receptors and TMPRSS2.

Glycosaminoglycans in the Pathogenesis of COVID-19

- Heparan sulfate proteoglycans are crucial for SARS-CoV-2 binding and infection 
- Low molecular weight heparins inhibit SARS-CoV-2 infection
- SARS-CoV-2 infection of epithelial cells is blocked by UF heparin and LMWH
- Heparan sulfates are required for transmission by primary dendritic cells
- Heparan sulfate proteoglycans Syndecan 1 and 4 are important for SARS-CoV-2 binding and infection.

 Heparan Sulfates and Low Molecular Weight Heparins in the Pathogenesis of COVID-19

While the mechanism remains unclear, it has been hypothesized that this type I sheddase transmembrane protein is involved in the pathogenesis of COVID-19.  Evidence that suggests that ADAM17 may be involved include:

- its known role in SARS-CoV viral uptake into host cells
- activating TNF‐α through cleave in cases of lung inflammation
- an association between high ADAM17 tissue expression and TNF‐α in an animal model of COPD, a known risk factor of COVID-19
- elevated ADAM17 gene expression in type II diabetic patients with diabetic low‐density lipoprotein (LDL)
- suggested contribution of ADAM17 to inflammation by stimulating IL-8 release
- proposed role in lung fibrosis and COPD through its ability to cleave IL-6R

ADAM17 in the Pathogenesis of COVID-19

A1AT is thought to be involved in the pathogenesis of COVID-19 because of its role as an innate immune system regulator, its function in lung homeostasis, and its potential relevance to the RAS system.  It may have a protective effect against SARS-CoV-2 infections by inhibiting the function of TMPRSS2 and TMPRSS2.  By inhibiting the serine protease ADAM17, which is involved in the viral uptake, the virus may be less efficient in infecting cells or may be prevented from infecting cells.  By inhibiting the sheddase ADAM17,  ACE2 cleavage would be controlled and may protect certain tissues from RAS system imbalance.  Additionally, inhibiting ADAM17 would prevent TNF‐α and IL6R cleavage which would have an anti-inflammatory effect which could ameliorate COVID-19 cytokine storm.  A1AT may also inhibit neutrophil elastase (NE), an enzyme that may be involved in SARS-CoV-2 S protein cleavage.  

A1AT in the Pathogenesis of COVID-19

In the innate immune response to SARS-CoV-2 infection, the host cells' pattern recognition receptors (PRR) recognise evolutionarily conserved molecular structures on the virus, called pathogen associated molecular patterns (PAMPS). When PAMPs bind to PRRs, the inflammatory response begins. PRRs activate several transcription factors to regulate the expression of genes responsible for inflammatory cytokine, interferon, chemokine, and adhesion molecule production.

Onset of Inflammatory Response in COVID-19

Researchers have found that COVID-19 virus is able to enter the body using another receptor, apart from ACE-2 receptor, called Neuropilin-1. This receptor is abundant in many tissues of the human body including respiratory tract, blood vessels and neurons. ACE-2 is expressed in very low levels on cell surfaces, the presence of Neuropilin in larger number may be one of the factor that attribute to its fast spread. 

Neuropilin-1 a receptor for COVID-19 entry 

Nsp stands for non-structural protein. This group of proteins are found in the SARS-CoV-2 virus. The SARS-CoV-2 virus contains Nsp1-16. These Nsp proteins help the virus replicate once it reaches its host cell. Some non-specific proteins play a larger role in cell entry and viral replication than others. 


Nsp proteins that play a significant role in the viral replication of the SARS-CoV-2 virus:

- Nsp1 (Non-structural protein 1)
- Nsp3 (Non-structural protein 3)
- Nsp9 (Non-structural protein 9)
- Nsp12 (Non-structural protein 12)
- Nsp15 (Non-structural protein 15)

Nsp's (Non-structural proteins) in the Pathogenesis of COVID-19

Other Type II Transmembrane Serine Proteases in the Pathogenesis of COVID-19

- tissue expression
- comparing expression to ACE2

TMPRSS2 Expression

TMPRSS2 is a serine protease involved in the cellular entry of SARS-CoV-2.  Its gene is regulated in part by upstream androgen receptors and has consequently been shown to be androgen-sensitive.  It may be possible to treat a SARS-CoV-2 infection using androgen deprivation therapy (ADT) or -anti-androgens which are currently used as prostate cancer therapies.  

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